This invention relates to a cell cultivating system to produce a target substance such as physiologically active substance and is useful to cultivate adhesive animal cells, particularly, the gene-transduced rodential cells which are prolific and require high concentration oxygen supply.
The well known processes of adhesive cell cultivation are generally classified into the suspending process (in homogeneous system) and the immobilizing process (in unhomogeneous system). Typical one of the suspending processes is so-called microcarrier process and, as the typical immobilizing process, the process is known, in which the cells are fixed on membrane or carrier.
(A) The microcarrier process in which the microcarrier is suspended in culture liquid to cultivate cells is disclosed, for example, in Japanese Patent Application Disclosure Gazette No. 1989-174376 proposed by inventors forming a part of the inventors of the present application and U.S. Pat. No. 4,904,601.
In such microcarrier processes of well known art, a vigorously stirred flow must be produced to assure uniform oxygen supply to the cells because the oxygen thus supplied is usually of a large quantity. However, a shearing force developed from the vigorous stirring often seriously damages the cells, causing the cell exfoliation.
(B) The well known immobilizing processes for cell cultivation include the process employing a mass of glass-wool as the cell matrix as disclosed in Japanese Patent Application Disclosure Gazette No. 1986-25483, the process employing a porous structure in the form of the steric screen as the cell matrix as disclosed in Japanese Patent Application Disclosure Gazette No. 1989-86870 and the process employing ceramic material as the cell matrix as disclosed in Japanese Patent Application Disclosure Gazette No. 1988-158715.
Of the well known immobilizing processes for cell cultivation, those employing, as the cell matrix, the mass of glass-wool, the porous structure in the form of the cylindrical screen and the regular-shaped ceramic particles, respectively, are disadvantageous commonly in that the culture medium passages are apt to be fixed and so-called channeling occurs. The term "channeling" used herein indicates a phenomenon that the cells deviating from the culture medium passages are destructed or inactivated.
To avoid such phenomenon resulting in closure or fixation of the culture medium passages, various researches and developments have been made on material and configuration of the carrier.
In addition to said channeling phenomenon, the prior art has encountered a serious problem such that continuous supply of the culture medium in a single direction often causes a significant gradient in concentrations of nutrient, pH and DO between inlet and outlet of the packed bed filled with the cell matrix and, in consequence, unevenness in cell proliferation as well as production of physiologically active substance.
Specifically, culture medium supply in a single direction often results in shortage of oxygen and increases waste product of the cells in the proximity of the outlet, adversely affecting the proliferation of the cells and production of the target substance. While accelerated circulation of the culture medium might solve such problem and thereby uniformalize the oxygen supply, a circulating velocity for the culture medium is practically limited to a predetermined linear velocity, because circulation of the culture medium through the packed bed at an extremely high linear velocity will result in the cell exfoliation caused by a shearing force of the stirred flow.
Moreover, the culture medium intrinsically tends to bubble since the culture medium is adequately supplied with oxygen and contains in itself the ingredients promoting the bubbling such as protein. Staying of such bubbles in the packed bed filled with the cell matrix also causes the channeling.
Though it is a commonly adopted technique to adjust dissolved oxygen concentration in a culture medium tank separately of the cell cultivating tank, it is also known to perform the adjustment of the extracellular environment within the cell cultivating tank. Specifically, Japanese Patent Application Disclosure Gazette No. 1985-9482 discloses such suspending process in which the culture medium is supplied together with air directly into the cell cultivating tank and the culture medium is separated from its supernatant liquid by the precipitator of cone type. With this process of prior art, however, the culture medium is directly mixed with air within the cell cultivating tank without relying on any particular mixing technique and therefore no sufficient mixing effect can be expected. Furthermore, supply of the culture medium is carried out merely by using a stirring propeller rotating in a constant direction, so uniform air supply is difficult.
While Japanese Patent Application Disclosure Gazette No. 1989-215276 discloses an example of the immobilizing process in which there are provided holding means for the cell matrix and the circulation circuit for the culture medium adjustment, the direction in which the culture medium is supplied is constant and therefore the above-mentioned problem is never solved by this process of prior art.